Intracellular neutral Lipid Droplets, their associated PAT proteins, and insulin resistance. Summary: Ectopic accumulation of lipid droplets in liver and muscle correlates with insulin resistance. Recent evidence indicates that lipid droplets are specialized organelles participating in lipid metabolism conferred by the activities of the lipid droplet-associated PAT protein family (Perilipin, ADRP, and Tip-47). Their occurrence at droplet surfaces suggests a role in lipid droplet biogenesis or metabolism. The functions of ubiquitously distributed ADRP and Tip47 remain unknown, but recent data suggest that they protect lipids from lipases. To determine the function of ADRP and Tip-47, we have down-regulated these two PAT proteins using siRNA treatment of AML-12 mouse liver cells. The absence of ADRP and Tip-47 at the surface of these lipid droplets was confirmed by immunocytochemistry, and no other PAT protein was identified at the surface of the lipid droplet. Lack of ADRP and Tip-47 resulted in a strong phenotype. When these cells were incubated in presence of 400 M of oleic acid, lipid droplet morphology was drastically altered, lipid droplet size increased by up to 10 fold and total number of lipid droplets per cell decreased by 50%. Lipid metabolism also was found perturbed: lipolysis rate increased by 3 fold. While NEFA uptake was not altered, incorporation of NEFA in the cellular triglyceride fraction was decreased by 50%. Further, the insulin signaling pathway was assessed by Akt phosphorylation in response to physiological insulin concentrations and found to be strongly attenuated in these cells p<0.001 as revealed by western blotting using Anti-phospho Akt antibodies. Other components in the insulin signaling pathway were similarly attenuated. In conclusion, our data support an important structural role for ADRP and Tip47 as surfactant proteins at the surface of the lipid droplet, packaging lipid in smaller units, protecting lipid droplet against endogenous lipase and facilitating triglyceride formation. These activities may have a role in the development of insulin resistance by liver tissue when lipid droplet accumulation occurs. Fatty acids are stored primarily as triacylglycerols (TAG) in intracellular lipid droplets, which are coated with member of the PAT protein family, which include the two broadly expressed proteins, ADFP and TIP47, and in tissue that oxidize fatty acids, LSDP5 as well. To date no cell devoid of PAT proteins has been reported. To determine the consequence of an absence of any PAT protein, we down-regulated the PAT proteins of AML12 liver cells with si mRNAs for ADFP and TIP47. Down regulation of either of these alone had not effect on lipid deposition or lipolysis, but the simultaneous down-regulation of both led to greatly increased droplet size and greatly increased lipolysis of stored TAG. This resulted in highly increased insulin resistance, as determined by attenuated phosphorylation of akt in response to insulin. Similarly, phosphorylation of a number of components of the insulin signaling pathway was altered. The rightward shift in the insulin dose-response curve was approximately 1-2 orders of magnitude, and the lipolytic rate mediated by ATGL was increased by nearly 3-fold. The insulin resistance was reversed upon down-regulation of the lipase, ATGL. Such data suggest that the unfettered release of Fatty acids into the cytosol was responsible for the insulin resistance. In conclusion, the data above suggest an important role of the PAT proteins is to sequester fatty acids as TAG in lipid droplets. Absent any PAT protein, the TAG are no longer protected from lipase action;consequently the fatty acids engage in a futile cycle of deposition in TAG, which are hydrolyzed and their Fatty acids rapidly released to the cytosol. These Fatty Acids render the cell resistant to insulin. A paper describing these results has been published in Diabetes. To assess the importance of PAT proteins in animal metabolism, the knock-out of any single PAT proteins is unlikely to yield productive information, because it is now known that the elimination of a single PAT proteins, such as ADFP, provokes the up-regulation of a compensatory PAT protein, such as TIP47. In muscle, it is likely that a loss of ADFP would elicit and up-regulation of LSDP5/OXPAT. To this end, we are producing mice with knock-outs of multiple PAT proteins, some total knock-outs and others, tissue-specific knock-outs. According to the revised nomenclature for the PAT protein, ADRP is now designated as perilipin 2(PLPN 2)and TIP47 as perilipin 3(PLPN3)and LSDP5 as PLPN 5. We now have PLPN2 null mice, which exhibits a 50-80% reduction in adipose tissue mass. By contrast, the PLPN 5 null mouse exhibits a 50% increase in adipose tissue mass. The PLPN3 null mouse colony is currently being expanded in order to produce animals with multiple knock-outs of the PAT proteins to produce double PLPN2/PLPN3 null mice, since these are the two most widely expressed lipid droplet coat proteins.